Metallothionein (MT) is a naturally occurring peptide, which is present in most cells of the mammalian body. There are many isoforms in humans, but these resolve into four classes; MT-I and MT-II which are expressed widely, MT-III which is mainly found in the brain, and MT-IV which is restricted to specific epithelial sites. MTs are intracellular proteins with occasional nuclear localisation, and although there are persistent reports of extracellular detection of MT, the prevailing dogma is fixed that their physiological role is within cells.
MTs are metal binding proteins (61-68 amino acids), which normally bind seven zinc ions, although zinc/copper mixtures have been reported. Some isoforms are rapidly induced in response to increases in zinc or copper levels, and also by a large number of hormones and cytokines, including glucocorticoids, interleukin 1 and 6, interferons and so on. Their exact physiological role is unclear. Early suggestions that they act to prevent accumulation of toxic levels of heavy metals are no longer favoured, and if their role is indeed in metal metabolism, it is more likely that they are involved in the intracellular homeostasis of zinc. However, MTs are efficient scavengers of free radicals and are able to protect DNA and other molecules from oxidation, suggesting that their function may be protective. MTs may be considered intracellular stress proteins which respond to a wide variety of stimuli.
It is relevant that MT-I/II knockout animals, and those which overexpress MT-I and MT-II are phenotypically normal, except for sensitivity and resistance, respectively, to some chemical and physical stresses.
Deficiency of MT-III, the “brain-specific” class of MT, has been implicated in the pathogenesis of Alzheimer's disease, although this finding has been strongly disputed. MT-III reduces neuronal survival, and the applicants have shown that, when MT-III is added to cultured neurons it reduces neurite sprouting. Exogenous MT-III appears to have an opposing effect to MT-IIA and it is expected that comparison of their structures will reveal strategies for designing analogues of both which have specific neurotrophic properties. It has been shown that exposure of rat brain lesions to MT-III causes vacuolisation, consistent with extensive neuronal death.
Metallothionein and Heavy Metals
There is a large body of literature on the relationship between metallothionein and heavy metals, particularly cadmium. MT was originally isolated as a cadmium-binding protein, and it is known that it acts as the major intracellular sink for this toxic metal. Hence, people exposed to cadmium in the workplace or through contaminated diet will have elevated metallothionein levels, particularly in the kidney. There is no question that MT acts to protect cells against cadmium, however it is not an effective agent, nor is it likely that this is the actual physiological role for the protein: it is likely an adventitious property derived from the chemical similarity between zinc and cadmium. One consequence of this is the numerous studies of the pharmacokinetics of metallothionein bound to heavy metals, following various routes of administration. Whilst cadmiummetallothionein is (not surprisingly) toxic, it is not believed that zinc or copper-metallothioneins will have significant metal-based toxicity at the concentrations employed in the studies below.
The applicants have examined the action of metallothionein proteins; including MT-IIA, a major human metallothionein of the MT-I/II class. The studies found that administration of metallothionein to cultured rat neurons increases neuronal survival and enhances the rate of axonal extension. Furthermore, in lesioned rat brains, metallothionein enhances regenerative axonal extension into the lesions and replacement of damaged tissue. Accordingly, the use of metallothionein as an active ingredient in neuronal therapy provides a novel method of stimulating neuronal growth and neuronal survival, a novel class of therapeutic agents and a novel method of treatment for a range of neuronally based disease states.
Moreover, metallothionein offers several practical advantages as a therapeutic agent.    1. It is a naturally occurring, non-toxic protein    2. It appears possible that intraperitoneally administered metallothionein can enter the CNS compartment, following physical trauma to the brain or spinal cord or breakdown of the blood-brain barrier due to other causes.    3. Metallothionein is not post-translationally modified and hence can be easily produced in bacterial or other expression systems    4. Metallothionein is a small peptide (61 amino acids for MT-IIA, 68 amino acids for human MT-III) and it is very likely that a novel analogue which is amenable to chemical synthesis can be designed.